Complex forming proteins are widespread in nature.
Fundamentally, these proteins can be assigned to the following groups:
antibodies which, with their antigen-binding sites, can bind specifically to the corresponding epitope of an antigen (which can also be another antibody)
protein complexes, such as occur, for example, in the activation of the clofting system or of the complement system
ligand receptor interactions of very different types, for example
of antigens or their epitopes with the Tcell or B-cell receptor
of growth factors, cytokines, chemokines, peptide hormones, steroid hormones and mediators with their respective receptors
of enzymes such as, for example, uPA or tPA with its receptor
of virus proteins with their respective cell receptor
adhesions between adhesion molecules
protein complexes in signal transmission, control of the cell cycle and control of the transcription of genes
Numerous examples of this are summarized in the literature, i.e. by Hardie et al., The Protein Kinase Facts Book I and II, Academic Press 1995, Callard et al., The Cytokine Facts Book, Academic Press 1994, Pigott et al., The Adhesion Molecule Facts Book, Academic Press 1994, Barclay et al., The Leukocyte Antigen Facts Book, Academic Press 1994, Watson et al., G-Protein Linked Receptor Facts Book, Academic Press 1994, Hesketh, The Oncogene Facts Book, Academic Press 1995, Leid et al., Cell 68, 377 (1992), Murre et al., Cell 58, 537 (1989), Bbeneza et al., Cell 61, 49 (1990), Brugge, Curr. Top. Microbiol. Immunolbiol. 123, 1 (1981), Callebaut et al., Proc. Natl. Acad. Sci. USA 89, 6270 (1992), Nadeau et al., J. Biol. Chem. 268, 1479 (1993), Burbach et al., Proc. Natl. Acad. Sci. USA 89, 8185 (1992), Hoffman et al., Science 252, 954 (1991), Stress-induced Proteins, M. L. Pardue, J. R. Feramisco and S. Lindquist Ed, A. R. Liss, New York (1989), Eukaryotic Transcription Factor, D. S. Lachman, Academic Press (1991), Transcriptional Regulations, Eds. S. McKnight and K. R. Yamamoto, CSHL Press, Cold Spring Harbor, New York (1992).
In some cases, the largely specific naturally occurring complexes formed, between at least two proteins, are utilized for the analysis or detection of proteins in the diagnosis of diseases (for this see EP 0 491 362 B1) and the binding partners of such proteins are used for the prophylaxis or therapy of disorders. If one partner of the particular protein complexes is available, with the aid of this partner the amount of the second or further component, be it, for example, complement or clotting factors, antigens, receptors or signal proteins, can be determined. Moreover, specific protein complexes are utilized in the search for small-molecule activators or inhibitors. In addition, purified antibodies or ligands for receptors, such as, for example, cytokines and peptide hormones, are administered to patients or animals for the prophylaxis or therapy of disorders.
In the case of these different application possibilities for proteins which form protein complexes, the specificity with which the respective proteins enter into complex formation and in addition the distribution of the respective proteins in the organism is of crucial importance. The lower the specificity, the more frequently nonspecific binding of a partner to foreign proteins will occur.
For example, nonspecific, i.e. undesired, formation of complexes with foreign protein partners is, as is known, the main problem of analysis and diagnosis using antibodies. Undesired formation of complexes with foreign protein partners can also be the cause of side effects in vivo, for example after injection of antibodies. In addition, the specific exclusive binding between two proteins is an essential prerequisite for the production and specific function of complex transcription factors, in particular of synthetic transcription factor complexes, such as have been described in the Patent Application EP-A 0 805 209.
There is thus a considerable need for novel, highly specific complex-forming proteins which do not react with foreign partners.
The invention relates to a complex formed from specifically complex-forming proteins which are not naturally occuring, wherein the following components are contained in the complex:
a) at least one ligand specific for a target structure,
b) at least one protein comprising a mutated dimerization domain, the mutated dimerization domain having been derived, i.e, obtained, by mutation of a naturally occurring dimerization domain, it being possible for this mutated dimerization domain to interact specifically, i.e., bind specifically, with component c) and the component b) being bonded covalently to the component a),
c) at least one protein comprising a mutated dimerization domain, the mutated dimerization domain having been derived, i.e., obtained, by mutation of a naturally occurring dimerization domain, it being possible for this mutated dimerization domain to interact specifically, i.e., bind specifically, with component b) and the component c) being bonded covalently to the component d), and
d) at least one effector.
According to the invention, a characteristic of the components b) and c) is that in naturally occurring peptides or proteins of identical or different type amino acids are replaced or inserted such that the peptides or proteins mutated in this way can complex virtually only exclusively with one another. A complexation of peptides or proteins mutated in this way with the corresponding nonmutated wild-type proteins or peptides does not occur, however. In this way, homodimers or heterodimers of mutated monomers (mutated peptides or proteins) can be formed.
The mutations in the binding domains of the identical or different proteins therefor have the purpose of preventing the ability for complex formation between an unmutated monomer and a mutated monomer of a pair of identical or different proteins or peptides which would complex in the unmutated state. At the same time, the mutations impart to such a pair of mutated proteins the ability to bind to one another with high specificity. The mutations thus occur in pairs, where one mutation is present in component b), the other in component c), i.e. such that a molecular interaction is possible between the respective amino acids of such a pair.
Amino acids according to the invention inserted into naturally occurring peptides or proteins can be, for example (the listing in pairs is intended to indicate the molecular interaction in the context of a dimeric protein complex):
The starting proteins for components b) and c) can be identical or different here.
In a preferred embodiment, the binding constant of a complex of two proteins according to the invention is at least KM=10xe2x88x927 mol lxe2x88x921, preferably at least KM=10xe2x88x928 mol lxe2x88x921.
Within the meaning of this invention, the following identical or unidentical partners (for the production of component b) or c) and with the aim of binding heterodimers), for example, are preferably mutated in their respective binding domain and these or the entire protein are used:
Those protein pairs are to be preferred which naturally have a helix-loop-helix/leucine zipper binding sequence.
The invention additionally relates to the very different uses of these complex-forming proteins according to the invention, for example
as multivalent protein complexes for prophylaxis and therapy
as multivalent ligands for vectors for gene therapy
as synthetic transcription factors for control of the expression of genes
and as diagnostic or analytical systems.
It is understood that these applications can either be accomplished in vivo or in vivo. Furthermore, it is understood that the gene therapy applications can be for either in vivo or ex vivo gene therapy, as these techniques are understood in the art.
In this context, the components a) and d) are selected depending on the chosen type of use.
The invention relates particularly to novel complex-forming proteins, wherein the amino acids mentioned have been inserted into proteins which naturally form homodimers or heterodimers, such that these proteins (components b) and c)) mutated in this way only form complexes with themselves (homodimers) or with the correspondingly mutated partner (heterodimers), but no longer with the naturally occurring, nonmutated starting proteins.
Novel complex-forming proteins containing the components a), b), c) and d) can be used in, for example, two embodiments.
In the first embodiment (see FIG. 1a), the component b) is a homo- (or hetero)multimer [component b)1-b)n], to which the corresponding identical (or different) components c) in each case bind, in each case as a monomer and in each case linked to the component d).
With this embodiment, many components d) (effectors) are bound to the target structure.
In the second embodiment (see FIG. 2), both the component b) and the component c) are multimers, only one or a few components d) being bound to the component c). Although with this embodiment only a few components d) (effectors) are bound to the target structure, the binding between the components c) and d) is extremely strong, which can increase the specificity of the binding of the components c) and d) to the target structure.
The invention relates to novel, complex-forming proteins consisting of the components a), b), c) and d), and also nucleic acid constructs which code for these complex-forming proteins. The invention likewise relates to complexes consisting of the components d), b), c) and d), i.e. the component a) is replaced by d), and nucleic acid constructs coding therefor.
In addition, the invention relates to a complex consisting of the components a), b), c) and a); i.e. the component d) is replaced by a), and nucleic acid constructs coding therefor.
The invention furthermore also relates to complex-forming proteins consisting of the components a), b), c) and d) or variants thereof described above, which additionally contain a fusogenic peptide, a translocation peptide or, between the components c) and d) or a) and b), a cleavage sequence for a protease, and nucleic acid constructs coding therefor.
All such nucleic acid constructs of this type can be introduced into bacteria, yeasts or mammalian cells with the aid of viral or nonviral vectors known to the person skilled in the art.
Cells of this type can be used for the preparation of the protein according to the invention or even administered for the purpose of the prophylaxis or therapy of an organism.
Nucleic acid constructs of this type, inserted in a vector, however, can also be administered directly to an organism for the purpose of prophylaxis or therapy.
The invention furthermore relates to the preparation of one of the abovementioned complexes, in which a protein of this complex is expressed with the aid of a nucleic acid construct coding therefor and a further protein of this complex, which is different from the first, is expressed with the aid of a nucleic acid construct coding therefor, and the expressed proteins are isolated and complexed with one another. In the case of complexes consisting of homodimers or homooligomers, the preparation is carried out without the expression of a second protein which is different from the first.
The invention further relates to:
A complex comprising the following components:
a) at least one ligand specific for a target structure;
b) at least one protein comprising a mutated dimerization domain obtained by mutation of a naturally occurring dimerization domain, wherein the mutated dimerization domain binds specifically with component c) and the component b) is covalently bonded to the component a);
c) at least one protein comprising a mutated dimerization domain obtained by mutation of a naturally occurring dimerization domain, wherein the mutated dimerization domain binds specifically with the component b) and the component c) is covalently bonded to component d); and
d) at least one effector;
wherein the components b) and c) are not naturally occurring proteins.
The above complex, wherein the component a) is replaced by the component d).
The above complex, wherein the component d) is replaced by the component a).
The above complex, which further comprises a fusogenic peptide or a translocalization peptide.
The above complex, which further comprises a cleavage sequence for a protease between the components c) and d) or a) and b).
The above complex, wherein the component a) is selected from the group consisting of: a growth factor, a cytokine, TNF, a chemokine, a peptide hormone, a mediator, a steroid hormone, a vitamin, a complement factor, a clotting factor, a kinin system factor, a fibrinolysis system factor, a plasmatic enzyme, a cell enzyme, a plasmatic enzyme inhibitor, a cell enzyme inhibitor, a virus coat protein, a cell receptor for the afore-mentioned molecules, an antibody, an antibody cleavage product, a DNA binding protein, a DNA binding domain of a transcription factor and an activation domain of a transcription factor.
The above complex, wherein the components b) and c) are obtained by mutating the naturally occurring dimerization domains of proteins which bind naturally to one another.
The above complex, wherein the naturally occurring dimerization domains of components b) and c) are selected from the group of naturally dimerizing partners consisting of:
The above complex, wherein the naturally dimerizing partners belong to the helix-loop-helix/leucine zipper family of proteins.
The above complex, wherein the mutated dimerization domains are obtained by inserting:
3-18 cysteines in the naturally occurring dimerization domain in at least one of the proteins of component b) and in at least one of the proteins of component c);
3-24 basic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component b) and 3-24 acidic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component c);
3-24 basic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component c) and 3-24 acidic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component b);
3-24 hydrophobic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component b) and 3-24 aromatic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component c);
3-24 hydrophobic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component c) and 3-24 aromatic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component b); and/or
3-24 aromatic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component b) and 3-24 aromatic amino acids in the naturally occurring dimerization domain in at least one of the proteins of component c).
The above complex, in which the components b) and c) are mutated binding domains of c-fos and c-jun, comprising the following mutations:
c-fos amino acid 167Exe2x86x92K
172Exe2x86x92K
181Exe2x86x92K
c-jun amino acid 283Kxe2x86x92E
288Kxe2x86x92E
302Kxe2x86x92E
The above complex, wherein the component d) is selected from the group consisting of: inhibitors of cell proliferation, apoptosis-inducing proteins, cytostatic proteins, cytotoxic proteins, coagulation-inducing factors, angiogenesis-inducing factors, angiogenesis-inhibiting factors, growth factors, cytokines, chemokines, interleukins, interferons, complement factors, clotting factors, fibrinolysis-inducing proteins, peptide hormones, mediators, bacterial proteins, receptors, viral antigens, parasitic antigens, tumor antigens, autoantigens, tissue antigens, adhesion molecules, antibodies, antibody cleavage products, enzymes for reacting with a signal-emitting component, enzymes for converting a precursor of an active substance into an active substance, fluorescent dyes, isotopes, metal-binding proteins, and a DNA-binding domain.
A method for treating diseased cells, wherein the disease results from inflammation, autoimmune diseases, defective formation of blood cells, nervous system damage, blood-clotting system disorders, blood circulation system disorders, tumor formation, viral infections, or bacterial infections, comprising administering the above complex to the cells.
A method for introducing a vector into a cell of an organism or cell culture, comprising binding the above complex to a viral or nonviral vector and introducing the vector into the cell of an organism or a cell culture in a cell-specific manner.
A method of detecting the presence or amount of a reactant in vitro or in vivo, comprising contacting the above complex with the reactant, either in vivo or in vitro, wherein the component a) binds to the reactant and the component d) emits a signal, such that the presence or amount of the reactant is detected by measuring the amount of the emitted signal.
A nucleic acid construct coding for a protein of the above complex.
The nucleic acid construct as above, coding for an activator subunit of an activator-responsive promoter unit.
A host cell comprising the nucleic acid construct as above.
The host cell as above, selected from the group consisting of a bacterium, a yeast and a mammalian cell.
A method of expressing a protein of the above complex, comprising expressing a nucleic acid encoding the protein in a host cell under conditions such that the protein is expressed in a detectable amount.
A method of producing a complex between the component b) and the component c) as in the above complex, comprising:
(a) expressing the at least one protein of the component b) by translating a nucleic acid construct encoding the protein;
(b) expressing the at least one protein of the component c) by translating a nucleic acid construct encoding the protein;
(c) isolating the proteins of steps (a) and (b);
(d) contacting the at least one protein of step a) with the at least one protein of step b) under conditions such that the proteins bind to one another.
The above complex, wherein the antibody cleavage product is selected from the group consisting of: F(ab)2, a single-chain Fv, a single-chain, a double antigen-binding molecule, and an Fc fragment.
The above complex, wherein the receptor is selected from the group consisting of receptors for: growth factors, cytokines, chemokines, interleukins, interferons, complement factors, clotting factors, fibrinolysis-inducing proteins, peptide hormones, steroid hormones, mediators, and virus coat proteins.
The above complex, wherein the antibody cleavage product is selected from the group consisting of: F(ab)2, Fab, single-chain Fv, and single-chain double antigen-binding proteins.
A vaccine comprising the above complex.
A complex comprising the following components:
a) at least one ligand specific for a target structure;
b) at least one protein comprising a mutated dimerization, wherein the mutated dimerization domain binds specifically with component c) and the component b) is covalently bonded to the component a);
c) at least one protein comprising a mutated dimerization domain, wherein the mutated dimerization domain binds specifically with the component b) and the component c) is covalently bonded to component d); and
d) at least one effector;
wherein the components b) and c) are not naturally occurring proteins.
The above complex, wherein at least one protein of component b) binds specifically with at least one protein of component c) with a binding constant of at least a KM of 10xe2x88x927 mol lxe2x88x921.